Biomedical subjects

Jens Pohl

Publications and source records attributed to Jens Pohl.

9 recordsLinked to original sources

PubMed2006

The effect of intracavernosal growth differentiation factor-5 therapy in a rat model of cavernosal nerve injury.

OBJECTIVE: To determine whether the intracavernosal application of growth differentiation factor-5 (GDF-5) influences nerve regeneration and erectile function after cavernosal nerve injury in a rat model. MATERIALS AND METHODS: Thirty-two male Sprague-Dawley rats were randomly divided into four equal groups: eight had a sham operation (uninjured controls), while 24 had bilateral cavernosal nerve crush. The crush-injury groups were treated at the time of injury with an impregnated collagen sponge implanted into the right corpus cavernosum. The sponge contained no GDF-5 (injured controls), 2 microg (low concentration), or 20 microg GDF-5 (high concentration). Erectile function was assessed by cavernosal nerve electrostimulation at 8 weeks. Midshaft penile tissue samples were histochemically evaluated for neuronal nitric oxide synthase (nNOS)-containing fibres in the dorsal penile nerve. RESULTS: There was no erectile dysfunction in the uninjured control group, as shown by a mean (sem) maximal increase in intracavernosal pressure (ICP) of 149.5 (17.0) cmH(2)O on stimulation. By comparison, the ICP decreased in the injured control group, by 21.3 (6.7) cmH(2)O. After cavernosal nerve injury, the recovery of erectile function was greatest in the low-concentration GDF-5 group; the maximum ICP increase was 40.8 (13.3) cmH(2)O, vs 24.3 (5.9) cmH(2)O for 20 microg GDF-5. Histologically, the low-concentration group had significantly more nNOS-containing nerve fibres, at 163 (24.7), than the high-concentration group, at 76 (17.3), or injured controls, at 67 (23.8). By contrast, the uninjured controls had a mean of 538 (40.6) nerve fibres in the dorsal nerve. CONCLUSION: Bilateral cavernosal nerve crush resulted in erectile dysfunction with accompanying neurological changes in the rat. The intracavernosal application of GDF-5 enhanced the recovery of erectile function and n-NOS nerve preservation, with a 2-microg dose giving the most promising results.

Animals↗

PubMed2006

Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity.

Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.

Alkaline Phosphatase↗

PubMed2005

Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2.

Here we describe 2 mutations in growth and differentiation factor 5 (GDF5) that alter receptor-binding affinities. They cause brachydactyly type A2 (L441P) and symphalangism (R438L), conditions previously associated with mutations in the GDF5 receptor bone morphogenetic protein receptor type 1b (BMPR1B) and the BMP antagonist NOGGIN, respectively. We expressed the mutant proteins in limb bud micromass culture and treated ATDC5 and C2C12 cells with recombinant GDF5. Our results indicated that the L441P mutant is almost inactive. The R438L mutant, in contrast, showed increased biological activity when compared with WT GDF5. Biosensor interaction analyses revealed loss of binding to BMPR1A and BMPR1B ectodomains for the L441P mutant, whereas the R438L mutant showed normal binding to BMPR1B but increased binding to BMPR1A, the receptor normally activated by BMP2. The binding to NOGGIN was normal for both mutants. Thus, the brachydactyly type A2 phenotype (L441P) is caused by inhibition of the ligand-receptor interaction, whereas the symphalangism phenotype (R438L) is caused by a loss of receptor-binding specificity, resulting in a gain of function by the acquisition of BMP2-like properties. The presented experiments have identified some of the main determinants of GDF5 receptor-binding specificity in vivo and open new prospects for generating antagonists and superagonists of GDF5.

Amino Acid Sequence↗

PubMed2005

Crystal structure of recombinant human growth and differentiation factor 5: evidence for interaction of the type I and type II receptor-binding sites.

The crystal structure of human growth differentiation factor 5 (GDF5) was solved at 2.4A resolution. The structure is very similar to the structure of bone morphogenetic factor 7 (BMP7) and consists of two banana-shaped monomers, linked via a disulfide bridge. The crystal packing of GDF5 is the same as the crystal packing of BMP7. This is highly unusual since only 25-30% of the crystal contacts involve identical residues. Analysis of the crystal packing revealed that residues of the type I receptor epitope are binding to residues of the type II receptor-binding epitope. The fact that for both BMP family members the type I and type II receptor-binding sites interact suggests that the complementary sites on the receptors may interact as well, suggesting a way how preformed receptor heterodimers may form, similar to the preformed receptors observed for the erythropoietin receptor and the BMP2 receptors.

Amino Acid Sequence↗

PubMed2004

Cartilage-derived morphogenetic protein-1 promotes the differentiation of mesenchymal stem cells into chondrocytes.

Mesenchymal stem cells (MSCs) are able to differentiate into many types of cells including chondrocytes. Transforming growth factor beta1 (TGF-beta1) is very important in the regulation of chondrogenesis. Since cartilage-derived morphogenetic protein-1 (CDMP-1) belongs to the TGF-beta superfamily, we tested whether CDMP-1 plays any role in the regulation of the differentiation of MSCs into chondrocytes using a high density pellet culture system. Based on the histological staining of glycosaminoglycan using toluidine blue dye-binding method we found that CDMP-1 could initiate chondrogenic differentiation of MSCs as did TGF-beta1. However, CDMP-1 was less stimulatory than TGF-beta1. The combination of CDMP-1 and TGF-beta1 synergically induced chondrogenesis of MSCs. This synergic chondrogenic effect of CDMP-1 together with TGF-beta1 was further confirmed by quantification of GAG using dimethylmethylene blue dye-binding assay and immunohistochemical analysis of the expression of cartilage-specific protein collagen II. This study may provide an improved induction approach using MSCs for repairing damaged cartilage.

Aborted Fetus↗

PubMed2004

Expression of growth differentiation factor-5 in the developing and adult rat brain.

Expression of the dopaminergic neurotrophin GDF-5 in developing rat ventral mesencephalon (VM) was found to begin at embryonic day (E) 12 and peak on E14, when dopaminergic neurones undergo terminal differentiation. In the adult rat, GDF-5 was found to be restricted to heart and brain, being expressed in many areas of the brain, including striatum and midbrain. This indicates a role for GDF-5 in the development and maintenance of dopaminergic neurones.

Age Factors↗

PubMed2004

Modulation of GDF5/BRI-b signalling through interaction with the tyrosine kinase receptor Ror2.

The brachydactylies are a group of inherited disorders of the hands characterized by shortened digits. Mutations in the tyrosine kinase receptor Ror2 cause brachydactyly type B (BDB). Mutations in GDF5, a member of the BMP/TGF-beta ligand family, cause brachydactyly type C (BDC) whereas mutations in the receptor for GDF5, BRI-b, cause brachydactyly type A2 (BDA2). There is considerable degree of phenotypic overlap between the subtypes BDB, BDC and BDA2. Here we demonstrate that all three components are involved in GDF5 induced regulation of chondrogenesis. We show that Ror2 (tyrosine kinase receptor) and BRI-b (serine/threonine kinase receptor) form a ligand independent heteromeric complex. The frizzled-like-CRD domain of Ror2 is required for this complex. Within that complex Ror2 gets transphosphorylated by BRI-b. We show that Ror2 modulates GDF5 signalling by inhibition of Smad1/5 signalling and by activating a Smad-independent pathway. Both pathways however, are needed for chondrogenic differentiation as demonstrated in ATDC5 cells. The functional interaction of Ror2 with GDF5 and BRI-b was genetically confirmed by the presence of epistatic effects in crosses of Ror2, BRI-b and Gdf5 deficient mice. These results indicate for the first time a direct interaction of Ser/Thr- and Tyr-Kinase receptors and provide evidence for modulation of the Smad-pathway and GDF5 triggered chondrogenesis.

Animals↗

PubMed2003

Expression of activins C and E induces apoptosis in human and rat hepatoma cells.

Activins C and E (homodimers of the betaC and betaE subunits), which are almost exclusively expressed in the liver, are members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. We examined their expression in three different hepatoma cell lines and found that, compared with normal liver or primary hepatocytes, human hepatoblastoma (HepG2), human hepatocellular carcinoma (Hep3B) and rat hepatoma (H4IIEC3) cells have either completely lost or drastically reduced the expression of activins C and E. In order to elucidate the biological function of these proteins we transiently transfected HepG2, Hep3B and H4IIEC3 cell lines with rat activin betaC or betaE cDNA to study the consequences of restoring activin expression in hepatoma cells. Transfection with activin betaA, a known inhibitor of hepatic DNA synthesis and inducer of apoptosis, served as a positive control. We found that transfection of the three cell lines with activin betaC or betaE, as well as with activin betaA, reduced the increase in cell number by up to 40% compared with cells transfected with a control plasmid. Co-culture with a CHO cell clone secreting activin C also inhibited HepG2 cell multiplication. Furthermore, the three hepatoma cell lines studied showed an enhanced rate of apoptosis and elevated levels of active caspases in response to activin transfection. These results indicate that activins C and E share the potential to induce apoptosis in liver derived cell lines with activin A and TGFbeta1.

Activins↗

PubMed2003

Bone augmentation using rhGDF-5-collagen composite.

The aim of this study was to evaluate the effectiveness of local application of growth differentiation factor-5 (GDF-5)-collagen composite on bone augmentation on the rat calvaria. GDF-5-collagen composite is made from recombinant human GDF-5 (rhGDF-5) and purified bovine type I atelocollagen. The GDF-5 solution was mixed with 0.3% atelocollagen acid solution, and the mixture was lyophilized. The spongy lyophilized material was pressed into the shape of a minidisk to make the GDF-5-collagen composite. The GDF-5-collagen composite contained 1, 10, or 100-microg rhGDF-5. The control collagen composite contained 0-microg rhGDF-5. The GDF-5-collagen composite or control collagen composite was inserted beneath the calvarial periosteum of 4-week-old rats. At 3 weeks after implantation, the implants containing 1-microg rhGDF-5 had mostly induced new bone formation on the cranial side. In the implants containing 10- microg rhGDF-5, bone formation had proceeded to the center of the GDF-5-collagen composite from the periosteal and the cranial sides, and bone marrow was seen focally. The augmented bone showed a connected trabecular structure with abundant vascularization. The implants containing 100-microg rhGDF-5 were nearly entirely replaced by new bone with bone marrow, and the augmented bone was firmly connected with the original bone. Neither cartilage nor bone formation was found in the control collagen composite. Thus, we conclude that the GDF-5-collagen composite may be a superior biomaterial for bone augmentation and this composite could be useful as a local osteoinductive device.

Animals↗